| Abstract [eng] |
T7 endonuclease I (T7E1) is a structure-selective endonuclease encoded by bacteriophage T7. T7E1 recognizes and catalyzes the cleavage of various branched DNA structures on both DNA strands – Holliday junctions, cruciform DNA, secondary structures of single-stranded DNA, etc. It can also recognize mismatches, insertions, or deletions in linear double-stranded DNA. T7E1 is used in gene synthesis for error correction, mutation detection in genome editing studies, and whole genome amplification experiments. According to the literature, T7 endonuclease I can recognize various DNA mismatches with different efficiency. Although the properties of the enzyme have been studied in quite some detail, a detailed scheme for the purification of the protein has not been yet published. In previous studies, several producer strains were created and the ones with the highest expression of recombinant T7 endonuclease I protein were selected. This work aims to create an efficient T7E1 protein purification scheme, analyze the cleavage of various heteroduplex substrates, and evaluate the potential use of the enzyme in a broader range of practical applications. During this work, the expression of the protein in different producer strains was evaluated, and a preliminary scheme for the purification of the T7E1 protein was created. After the protein was purified, the physical and functional purity was evaluated, as well as the activity of the protein using different constructed substrates. The properties of the T7 endonuclease I protein were analyzed, such as specific activity, thermal inactivation, thermal stability, activity over time, and the possibilities of practical use were evaluated as well. |
